Figure 3.

Exocytosis Defects in Wild-Type and gef1Δ Cells after Orb6 Inhibition

(A) Video time points of mCherry-Bgs4 after combining actin depolymerization by latrunculin A (LatA) with Orb6 inhibition. Prior disruption of endocytosis by LatA prevents loss of Bgs4 from cell tips after Orb6 inhibition (compare with Figure 2A). Video S6 shows more cells, including this cell, together with gef1Δ cells treated in the same way.

(B) Secretion of acid phosphatase (ACP) activity into culture medium in indicated strains after treatment with methanol (left) or 30 μM 3-BrB-PP1 (right). Graphs show mean ACP activity normalized to cell density from replicate experiments. Error bars show SEM.

(C) Fluorescence recovery after photobleaching (FRAP) of plasma membrane t-SNARE GFP-Psy1 at cell tips in orb6-as2 cells under the indicated conditions. The top-left diagram shows the target bleaching zone (magenta), line scan along the plasma membrane (green), and bleached portion of the plasma membrane (orange). The top images are from representative experiments. The bottom images are corresponding kymographs based on the line scan and bleached portion shown in the diagram.

(D) Quantification of FRAP from experiments as in (C) (see STAR Methods). Symbols indicate mean values. Error bars indicate SD. n indicates the number of cells analyzed.

Scale bars, 10 μm. The imaging experiment for (A) was performed once. For (B), four biological replicates were performed for wild-type and orb6-as2 cells, and two biological replicates were performed for orb6-as2 gef1Δ cells. Two biological replicates of FRAP imaging were performed for (C).